Device for isoelectric focussing

ABSTRACT

A device for separating one or more components in a sample is disclosed, the device including: (a) a first planar member ( 1 ) having a channel ( 2 ) along which a sample may be loaded and component(s) thereof focussed isoelectrically; and (b) means for exposing the channel ( 2 ) along at least a portion of its length and thereby exposing the sample or component(s) therewithin. The means for exposing the channel ( 2 ) may include a cover plate ( 7 ). A method of analysing a molecule is also provided, the method including: (a) providing an elongate open channel ( 2 ); (b) introducing a plurality of molecules into the elongate open channel ( 2 ); (c) separating molecules along the elongate open channel ( 2 ) according to their isoelectric points; (d) accessing a molecule in the elongate open channel ( 2 ) and analysing it. A use of the device in combination with a mass spectrometer, preferably a MALDI-TOF unit, for analysis of a protein is also disclosed.

FIELD

The present invention relates to a device and method for separatingmolecules, in particular macromolecules such as proteins. In particular,the invention relates to a device capable of charge-based separation ofproteins.

BACKGROUND

The separation of molecules in a complex mixture is often desired forvarious purposes. For example, a multitude of proteins exist within acellular environment, and in order to aid characterisation, it is oftennecessary to separate these proteins from each other. Various separationtechniques have been developed, each of which rely on one or morediffering properties of the proteins to separate them from each other.

For example, sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) separates proteins according to their size. In SDS-PAGE,proteins are denatured and solubilised in a SDS buffer, negativelycharged SDS molecules bind to the protein, with more molecules bindingto larger proteins. On application of an electric field, proteinsmigrate in a polyactylamide gel according to their charge (and hencesize). The electric field is turned off to immobilise the proteinswithin the gel. We can refer to techniques such as SDS-PAGE as “singledimension” separation, as separation is based on only one property ofthe protein (in this case mass).

The analysis of complex mixtures, however, often requires more than oneseparation process in order to resolve all the components present in asample. It is for this reason that two dimensional (2D) separationschemes have been devised.

Two dimensional separation techniques make use of two properties of theproteins for separation. Separation is carried out in one dimension byuse of a first property, and then a second dimension (which is generallyorthogonal or perpendicular to the first dimension) by means of a secondproperty. When constructing a successful 2D system several criteria needto be addressed. For example, the two techniques should base theirrespective separations on as different a means as possible. Doing sowill reduce the amount of redundant information contained in the 2Ddataset. 2D techniques are advantageous as they provide higherresolution. For example, they may be able to resolve several differentproteins which differ only marginally in mass, but have differentcharges (such as in the case of differentially phosphorylated proteins).

Two dimensional polyacrylamide gel electrophoresis (2-D PAGE) is apopular and currently favoured technique for protein separation(Anderson N. G., Anderson N. L., Electrophoresis 1996; 17, 443453).Proteins are first subjected to isoelectric focusing (IEF) in animmobilized pH gradient in the slab gel format to separate proteinsaccording to their charge (pI values), a step which typically takesabout 6-8 hours. Then, the IEF gel is placed on top of a gradient geland electrophoresed in the presence of SDS to separate proteins based ontheir molecular mass. The separated proteins are stained forvisualization, interested bands are excised and digested with proteasefollowed by peptide finger printing by mass spectrometry for proteinidentification (Shevchenko, A., Jensen, 0. N., Podtelejnikov, A. V.,Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Boucherie, H., Mann,M., Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445; Jensen, O. N.,Larsen, M. R., Roepstorff, P., PROTEINS 1998, 74-89 Suppl. 2).

2-D PAGE is the current technology of choice for large scale proteomicsanalysis because 2-D PAGE is the highest resolution method for proteinseparation and the pattern of proteins in the 2-D map is related to theproperties of proteins, namely isoelectric point in first dimension andmolecular mass in the second dimension. Therefore, the positions ofproteins in 2-D map correspond to their chemical and physicalproperties. These properties can be used to identify and characterizethe proteins. 2-D PAGE has been used to analyze human plasma proteins,and the pI and molecular weight of proteins can be used for detectionand diagnosis of diseases in clinical analysis (Rasmussen, R K., Ji, H.,Eddes, J. S., Moritz, R L., Reid, G. E., Simpson, R. J., Dorow, D. S.,Electrophoresis 1997, 18, 588-598).

However, 2-D PAGE is a labour intensive procedure and difficult toautomate, it also suffers from its limitations in sensitivity anddynamic range of detection. Virtual 2-D gel electrophoresis has recentlybeen developed (Ogorzalek-Loo, R. R., Cavalcoli, J. D., VanBogelen, RA., Mitchell, C., Loo, J. A., Moldover, B., Andrews, P. C., Anal. Chem.2001, 73, 40634070), where mass spectrometry replaces the size-basedseparation of SDS-PAGE in the second dimension. It has been shown thatthis technology is more sensitive than 2-D PAGE. However, the firstdimension of separation is still performed in polyacrylamide gel,limiting the potential for high throughput analysis.

Mass spectrometry (MS) is an important analytical technique formolecular structure characterization because of its high specificity,sensitivity and speed (McLafferty, F. W., Science 1981, 214, 280287).Techniques such as electrospray ionization (ESI), described in Fenn, J.B., Mann, M., Meng, C. K., Wong, S. F., Whitehouse, C. M., Science 1989,246, 64-71, and matrix-assisted laser desorption/ionization (MALDI),described in Karas, M., Hillenkamp, F., Anal. Chem 1988, 60, 2299-2301,have greatly extended the capacity of mass spectrometry to studynon-volatile and labile biomolecules. A variety of micro-separationtechniques have been exploited to interface to mass spectrometry formolecular identification. The interface of microcolumn separation toelectrospray ionization mass spectrometry is the most common system(Tomer, K. B., Chem. Rev. 2001, 101, 297-328), and is described forexample in U.S. Pat. No. 5,993,633. Other techniques involve for exampledepositing effluent from capillary electrophoretic separation on a MALDItarget plate (Minarik M., Foret F., Karger B. L., Electrophoresis 2000,21, 247-254).

In capillary zone electrophoresis (CZE) a sample is dissolved in abuffer and the sample is injected at one end of a separation capillaryor channel. Ie separation capillary or channel may also be loaded with auniform buffer solution, and a sample injected at one end. A constantvoltage potential is applied along the separation channel so that ionsmove at rates corresponding to their electrophoretic mobilities. Sincedifferent ionic species have different charge-to-mass ratios, theyseparate as they migrate along the channel. Liu et al (2001, Anal. Chem.73, 2147-2151) describe a 2-dimensional separation system, which couplescapillary zone electrophoresis (CE or CZE) with MALDI. Separation isfirst performed in open microchannels manufactured on glass microchips.Samples are introduced at one end of the channel, and separated. Themicrochips are then transferred to a MALDI source after evaporation ofsolvent. Separation in the first dimension occurs by a combination ofelectrophoresis and electroosmosis, and electroosmotic movement ofpeptides and oligosaccharides is demonstrated.

Electroosmosis, or electroendosmosis, is a bulk flow phenomenon whichaffects separation during capillary electrophoresis, particularly inglass channels. The velocity of an analyte in capillary electrophoresisdepends not only on the forces applied by the electrical potential, butalso upon the rate of endoosmotic flow (EOF) within the channel.Endoosmotic flow is observed when an electric field is applied to asolution contained in a capillary with fixed charges on the capillarywall, for example, a glass capillary wall. Typically, charged sites arecreated by ionization of silanol groups on the inner surface of thefused silica. Silanols are weakly acidic, and ionize at pH vales aboveabout pH 3. Hydrated cations in solution associate with ionized SiO⁻groups to from an electrical double layer, a static inner layer close tothe surface (also known as the Stern Layer) and a mobile outer layer(also termed the Helmholtz plane). Upon application of an electricfield, hydrated cations in the outer layer move towards the cathode,creating a new flow of the bulk liquid in the capillary in the samedirection. The rate of movement is dependent on the field strength andthe charge density of the capillary wall. The population of chargedsilanols is a function of the pH of the medium, so that the magnitude ofthe EOF increases directly with pH until all available silanols arefully oxidised. Electroosmosis is described in further detail in Wehr,T., Rodriguez-Diaz, R, Zhu, M., Capillary Electrophoresis of Proteins,Marcel Dekker, Inc., New York, 1999.

Capillary isoelectric focussing (CIEF) is an equilibrium-based method ofseparation that depends on a pH gradient created by carrier ampholyte.Proteins move under an electric field to their pI points where theycarry zero charge and are focused. Therefore, separation andconcentration occur at the same time. The concentration of proteins atthe focused zone can be increased by 100-500 times relative to thestarting solution because the same protein in the whole capillary isfocused on a single spot.

Single point detection techniques, such as laser induced fluorescenceand ESI-MS, have been employed to detect the separated proteins afterCIEF. Focused protein zones need to be mobilized in order to passthrough the detection point at the end of the tube (Rodriguez, R., Zhu,M., Wehr, T., J Chromatogr. A 1997, 772, 145-160).

MAILDI, such as MALDI-MS and MALDI-TOF are important techniques formeasuring large molecular masses accurately and studying protein-ligandinteractions, but successful interfacing with chromatography, inparticular, capillary electrophoresis, has yet to be successfullyachieved. The problem of interfacing CIEF MALDI-MS is because thefocused protein zone inside the capillary cannot be reached directly.Therefore, the contents of the capillary need to be mobilized out of thecapillary and deposited into an appropriate surface for subsequenceMALDI ionization. This mobilization step degrades the resolution,increases the analysis time, and distorts the pH gradient. Hence, theresult reproducibility is poor.

SUMMARY

According to a first aspect of the present invention, we provide anisoelectric focussing (IEF) module comprising: (a) a first planar memberhaving a channel along which a sample may be loaded and a component orcomponents thereof focussed isoelectrically; and (b) means for exposingthe channel along at least a portion of its length and thereby exposingthe sample or component(s) therewithin.

Preferably, the sample or component(s) are accessible along the exposedchannel at substantially the positions at which they are focussed.Preferably, the channel comprises an open channel which is exposed alongat least a portion of its length. Preferably, the channel comprises alinear groove formed on a surface of the first planar member.

In preferred embodiments, the channel comprises a microchannel orcapillary channel. Preferably, the microchannel or capillary channel ismicrofabricated on the first planar member.

The channel may have a width of between 1 to 500 micrometres, morepreferably between 50 to 350 micrometres, more preferably between 50 to350 micrometres, most preferably about 150 micrometers or about 175micrometres.

The first planar member may be formed from a material selected from thegroup consisting of: plastics, polymers, ceramic, glass or composite.Preferably, at least one wall of the channel comprisespoly(methylmethacrylate) (PMMA) or polycarbonate. Preferably, the firstplanar is coated or derivatised to reduce surface charge and therebyminimise electroosmotic flow (EOF).

The module may comprise reservoirs for electrolyte, the reservoirs beingin electrical connection with the channel. Preferably, the reservoirsare formed on the first planar member adjacent to each end of thechannel.

In some embodiments, the module further comprises a lid being a secondplanar member, which reduces evaporation of the sample in the channel.Preferably, the lid comprises an elongate recess on its inner face, therecess being positioned such that when the lid is mated with the firstplanar member, no substantial leakage of sample contained in the channeloccurs.

Preferably, the length and width of the recess are at least as great asa channel in the first planar member. In such embodiments, thereservoirs are preferably disposed on the lid.

Preferably, the module comprises means for electrical connection betweenthe channel and the reservoir, but preventing substantial mixing ofsample and electrolyte. The means may comprise a semi-permeablemembrane, agarose, acrylamide, agar, or a gel plug.

In highly preferred embodiments, the module comprises a plurality ofchannels in substantially parallel orientation.

In certain embodiments, the channel or channels comprises a closedchannel(s) and the means for exposing the channel(s) comprises lines ofweakness enabling fracture along a longitudinal plane of the channel(s).

Preferably, the module comprises a translational stage on which ismounted the first planar member.

There is provided, according to a second aspect of the presentinvention, an apparatus for separating one or more components in asample, the apparatus comprising an isoelectric focussing (IEF) moduleas set out in the first aspect of the invention, together with a modulecapable of separating isoelectrically focussed components according totheir respective masses.

Preferably, the mass separation module comprises a module for massspectrometry. Preferably, the mass separation module comprises a matrixassisted laser desorption/ionisation mass spectrometry (MALDI-MS)module, preferably, a matrix assisted laser desorption/ionisation-timeof flight (MALDI-TOF) module.

We provide, according to a third aspect of the present invention, amethod of separating one or more components in a sample, the methodcomprising the steps of: (a) providing an isoelectric focussing (IEF)module comprising a first planar member having a channel; (b) loadingthe channel with a sample; (c) isoelectrically focussing a component orcomponents of the sample along the channel; and (d) exposing the channelalong at least a portion of its length and thereby exposing the sampleor component(s) therewithin.

The method may comprise one or more features as defined in the preferredembodiments of the first aspect of the invention.

The method may further comprise the step of: (e) accessing one or morecomponents in the open channel and analysing it. Preferably, the or eachcomponent is analysed by mass spectrometry, preferably by MALDI-MS, morepreferably by MALDI-TOF mass spectrometry.

Preferably, the sample comprises a MALDI matrix. Preferably, a MALDImatrix is added to the sample subsequent to isoelectric focussing.Preferably, the MALDI matrix is selected from the group consisting of:Cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxy benzoic acid (DHB),Alpha CCA, Sinapinic Acid (SA), 3-hydroxypicolinic acid (HPA), IAA(Na⁺), 2-(4-Hydroxyphenylazo)benzoic acid HABA (Na⁺), Dithranol (Na⁺),Retinoic Acid (Na⁺), Succinic acid, 2,6-Dihydroxyacetophenone, FerulicAcid, Caffeic acid, Glycerol and 4-Nitroaniline.

As a fourth aspect of the present invention, there is provided a methodof analysing a molecule, the method comprising: (a) providing anelongate open channel; (b) introducing a plurality of molecules into theelongate open channel; (c) separating molecules along the elongate openchannel according to their isoelectric points; (d) accessing a moleculein the elongate open channel and analysing it.

We provide, according to a fifth aspect of the present invention, anapparatus for isoelectric focussing (IEF) of molecules, the apparatuscomprising an elongate channel, in which the elongate channel is openalong at least a portion of its length to enable separated molecules tobe accessed.

The present invention, in a sixth aspect, provides a CIEF-MALDIapparatus.

In a seventh aspect of the present invention, there is provided a kitcomprising a module or apparatus as described above, together with asample comprising proteins to be analysed.

According to an eighth aspect of the present invention, we provide useof an isoelectric focussing module as described above, in combinationwith a mass spectrometer, preferably a MALDI-MS unit, more preferably aMALDI-TOF unit, for analysis of a protein. Preferably, such use is forproteome analysis.

We provide, according to a ninth aspect of the invention, a means forinterfacing a capillary isoelectric focussing (CIEF) apparatus and aMALDI-MS apparatus (preferably a MALDI-TOF apparatus), the interfacemeans comprising a channel along which isoelectric focussing is carriedout, and means for exposing said channel along at least a portion of itslength and thereby exposing the sample or component(s) therewithin.

Preferably, the interface comprises an open channel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a plan view of a first embodiment of aseparation device as described here, comprising a single open channelwith reservoirs “in cis” (i.e., on the same substrate as the openchannels). 1: substrate, 2: open channel, 3 and 4: anolyte and catholytereservoirs (electrolyte reservoirs), 31: agarose gel plug.

FIG. 1B is a diagram showing a longitudinal cross section of theembodiment of the separation device shown in FIG. 1A.

FIGS. 2A to 2D are diagrams showing transverse cross sections ofembodiments of the separation device comprising a single open channel,illustrating various configurations of the open channel. FIG. 2A shows achannel with straight walls and base. FIG. 2B shows a “U” shaped channelwith straight walls and a curved base. FIG. 2C shows a “U” shapedchannel with curved walls and a straight base. FIG. 2D shows a “U”shaped channel with curved walls and a curved base.

FIG. 3 is a diagram showing a longitudinal cross section of theembodiment of the separation device shown in FIG. 1A, mounted on astage. The stage may comprise for example an XY-translation stage forMALDI. 5: stage, 6: laser beam.

FIGS. 4A to 4C are diagrams showing a second embodiment of a separationdevice as described here, comprising multiple open channels withintegral reservoirs. FIG. 4A shows a plan view and FIG. 4B shows atransverse cross section of the separation device. FIG. 4C shows alongitudinal cross section of the separation device mounted on a stage,for example an XY-translation stage for MALDI. 1, 2, 3, 31, 4, 5, 6 areas described in legend to FIG. 1A and FIG. 3.

FIGS. 5A to 5C are diagrams showing a third embodiment of a separationdevice as described here, comprising a single open channel andreservoirs “in trans” (i.e., not on the same substrate as the openchannels). FIG. 5A: plan view of separation device with lid detached.FIG. 5B: plan view of device with lid in place. 7: lid, 8: recess inlid, shown in outline (dashed lines).

FIGS. 6A and 6B are diagrams showing a transverse section of the thirdembodiment of FIG. 5 along a plane of the reservoir. FIG. 6A: openconfiguration; with lid removed. FIG. 6B: closed configuration, with lidin place.

FIGS. 7A to 7C are diagrams showing a fourth embodiment of a separationdevice as described here, comprising multiple open channels andreservoirs “in trans”, in which the reservoirs are on a separate piecefrom the open channels. FIG. 7A: plan view of separation device withoutlid. FIG. 7B: plan view of separation device covered with lid. Recesses(8) in lid are shown in outline.

FIGS. 8A and 8B are diagrams showing a transverse section of the fourthembodiment of FIG. 7 along a plane of the reservoirs. FIG. 8A: openconfiguration, with lid removed. FIG. 8B: closed configuration, with lidin place.

FIG. 9 is a composite photograph showing isoelectric focussing ofmyoglobin. Top lane: t=0, bottom lane: t=end of experiment. Arrow showsdirection of time.

FIG. 10 is a photograph showing the separation device mounted on anadaptor for attachment to a MALDI sample plate. 9: adaptor.

FIG. 11 is a graph showing MALDI TOF-MS of myoglobin from a focussedzone in the separation device. X-axis: mass to charge (m/z) ratio,Y-axis: intensity.

FIGS. 12A and 12B are photographs showing separation of whole porcineliver proteins. FIG. 12A: open channel with visible focussed protein(arrow), the adjacent ruler shows a scale and is intended to estimatethe pI value. FIG. 12B: enlargement of FIG. 12A showing three visibleprotein spots at about 8 cm, indicated by arrows.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J.E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A:Synthesis and Physical Analysis of DNA Methods in Enzymology, AcademicPress. An extensive review of 2-D PAGE techniques, and their applicationin proteome analysis, is provided by Andrew J. Link, 2-D ProteomeAnalysis Protocols, Vol. 112, Humana Press ISBN: 0896035247. Each ofthese general texts is herein incorporated by reference.

DETAILED DESCRIPTION

We disclose a module/apparatus which separates proteins usingisoelectric focussing, and which is adapted for easy interfacing withmass spectrometry, in particular MALDI mass spectrometry (MALD-MS). Inparticular, the module is adapted for easy interface with MALDI-TOF. Inparticular, our module/apparatus employs 2D separation using charge in afirst dimension (isoelectric focussing), and mass in the second (MALDI,preferably MALDI-TOF).

The apparatus/module and method described here enables rapid andaccurate focussing of components of a sample, in particular, proteincomponents of the sample, along a channel, and enabling access to these.This is achieved by providing means for exposing the channel along atleast a portion of its length, preferably the whole or substantially thewhole of its length. Specific embodiments of such a device and methodare described in further detail below. Embodiments where the channel is“open” are preferred, and provide random access to any target proteinalong the microchannel. The target protein may be extracted, or may beanalysed in situ. The target protein may be removed for analysis, forexample, by interfacing the channel or microchannel to a massspectrometry apparatus. Use of specific MS apparatus, such as MALDI orMALDI-TOF, is preferred.

The module, apparatus and method described here are therefore capable ofdetecting components in the sample, in particular, determining one ormore properties of the or each component. In preferred embodiments, themodule, apparatus and method described here is used for proteomeanalysis, i.e., analysing the protein components of a cell. The module,apparatus and method described here may also suitably be used fordetection of one or more disease associated proteins in a sample from anindividual. Such detection may be used as, or as a means to determine, adiagnosis of a disease.

Samples and Components

The method and apparatus described here is suitable for separating oneor more components from a sample, which is typically a mixture ofcomponents. The sample may be a complex mixture, comprising hundreds orthousands of components. The components may be uniform in nature, butpreferably are not In preferable aspects, two or more of the componentsmay be distinguished by one or more properties, for example, charge,mass, etc.

Samples which are suitable for isoelectric focussing using our module orapparatus may therefore include different types. In particular, ourmethods and apparatus are suitable for separation and analysis ofcomplex samples, for example, cell extracts. Cell and tissue extractsmay be prepared by any means known in the art.

The samples may comprise simple molecules, complex molecules, or anymixture of these. They may comprise proteins, carbohydrates, nucleicacids, DNA, RNA, etc. Preferably, at least one of the components of thesample comprises an amphoteric molecule, such as a protein.

The sample may comprise one or more of the following: a protein, apeptide, a polypeptide, an amino acid, an oligonucleotide or modifiedoligonucleotide, an antisense oligonucleotide or modified antisenseoligonucleotide, cDNA, genomic DNA, an artificial or natural chromosome(e.g. a yeast artificial chromosome) or a part thereof, RNA, includingmRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virusor virus-like particles; a nucleotide or ribonucleotide or syntheticanalogue thereof, which may be modified or unmodified; an amino acid oranalogue thereof, which may be modified or unmodified; a non-peptide(e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate,etc.

Protein Containing Samples

Our method and device may be used to analyse any sample, in particularprotein containing samples. In preferred embodiments, the methods andapparatus described here is suitable for separating samples comprisingproteins. Preferably, the molecules which are isoelectrically focussedand/or analysed comprise proteins.

The proteins may preferably be human proteins, or animal proteins,mammalian proteins or bacterial or other microorganism proteins. Theproteins may be native proteins, or denatured proteins. They maycomprise wild type proteins, or mutated proteins, whether natural or manmade. They may comprise post translational modifications, for example,any one or more of ADP-ribosylation, ubiquitination, glycosylation,prenylation (fatty acylation), sentrinization, phosphorylation, etc. Theproteins may comprise one or more post-translationally modified groupssuch as methyl, phosphate, ubiquitin, glycosyl, fatty acyl, sentrin orADP-ribosyl moiety. Such modifications are described for example in WO00/50896, WO 00/50635, WO 00/50631, WO 00/50630 and GB2342652. Theprotein may be an isoform, and the sample may in particular comprise oneor more protein isoforms.

The proteins may comprise recombinantly expressed proteins. Methods ofproducing recombinant proteins, methods of expression, vectors, andhosts suitable for expression are well known in the art.

Disease Associated Proteins

In preferred embodiments, the protein or proteins which is detected oranalysed comprises a disease associated protein. By this term we mean aprotein whose presence in a cell, tissue or organ of an individual isindicative of a disease state of the cell, tissue or organ. In preferredaspects, the protein is a flag or marker of a pathological condition.The protein may be a causative agent of the disease state, or it may nothave any causative effect The protein may be a “downstream” indicator ofdisease. The disease associated protein may be indicative of thepresence of the disease, or susceptibility to the disease, in anindividual.

It will be appreciated that the disease associated protein itself neednot be detected, and that any nucleic acid encoding it, for example, adisease associated-DNA, -mRNA, -gene, -allele, etc may be detected.

The disease may comprise any known disease, which affects humans oranimals. The disease may in particular comprise infections such asbacterial, fungal, protozoan and viral infections, particularlyinfections caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity;anorexia; bulimia; asthma; Parkinson's disease; thrombosis; acute heartfailure; hypotension; hypertension; erectile dysfunction; urinaryretention; metabolic bone diseases such as osteoporisis and osteopetrosis; angina pectoris; myocardial infarction; ulcers; asthma;allergies; rheumatoid arthritis; inflammatory bowel disease; irritablebowel syndrome benign prostatic hypertrophy, and psychotic andneurological disorders, including anxiety, schizophrenia, manicdepression, delirium, dementia, severe mental retardation anddyskinesias, such as Huntington's disease or Gilles dela Tourett'ssyndrome. Inflammatory diseases such as psoriasis, acne, eczema, etc arealso included.

Preferred diseases include those which afflict or threaten first worldpopulations, such as AIDS, cancer, Alzheimers disease, Parkinsons, CJD,etc.

The disease associated protein for a specific disease may be one whichhas previously been determined (i.e., a known disease associatedprotein), or it may be unknown. In the latter case, the methods,apparatus and module described here may suitably be utilised todetermine the unknown disease associated protein.

A sample from a diseased individual is taken, and separated and analysedas described. One or more profiles may be generated; these may comprisefor example, an isoelectric focussing profile (the disposition of thevarious proteins along the channel), or preferably a mass spectrometryprofile. The mass spectrometry profile will include information on themolecular weights of the proteins present in the disease sample. Thedisease profile is then compared with a relevant profile generated froma normal (i.e, undiseased) individual.

Any differences in the profile indicate differences in the proteincompositions of a normal versus a diseased individual. Such differencesmay provide markers for disease, and be used as putative diseaseassociated proteins. They may be detected in other individuals asdescribed to determine the presence of a disease, or susceptibilitythereto.

Detection of such a disease associated protein in a cell, organ, etc,using the methods and apparatus described here may be used as an aid todiagnosis of the disease For certain diseases, such detection may beused as a direct diagnosis of the disease. Appropriate treatment maythen be administered to the individual or patient in question.

Isoelectric Focussing

The module makes use of isoelectric focussing along a channel,preferably a narrow channel.

The term “isoelectric focussing” also known as IEF or electrofocusing,should be understood to refer to a technique in which solutes ofdifferent isoelectric points are caused to form stationary bands in anelectric field, which is superimposed on a (stable) pH gradient, the pHincreasing from the anode to the cathode. Preferably, the pH gradient ismost conveniently formed by electrolysing a solution containing amixture of carrier ampholytes of low molecular mass and slightlydiffering isoelectric points, each of which will move to its isoelectricregion in the electric field and remain there.

In further detail, isoelectric focusing (IEF) is an electrophoretictechnique that adds a pH gradient to the buffer solution and togetherwith the electric field focuses most biological materials that areamphoteric. Amphoteric biomaterials such as proteins, peptides, nucleicacids, viruses, and some living cells are positively charged in acidicmedia and negatively charged in basic media. During IEF, these materialsmigrate in the pre-established pH gradient to their isoelectric pointwhere they have no net charge and form stable, narrow zones. Isoelectricfocusing yields such high resolution bands because any amphotericbiomaterial which moves away from its isoelectric point due to diffusionor fluid movement will be returned by the combined action of the pHgradient and electric field. The focusing process thus purifies andconcentrates sample into bands that are relatively stable.

Isoelectric focussing is an electrophoretic process. “Electrophoretic”separations refers to the migration of particles or macromoleculeshaving a net electric charge where said migration is influenced by anelectric field. Accordingly electrophoretic separations contemplated foruse in the apparatus and method described here include separationsperformed in channels packed with gels (such as polyacrylamide, agaroseand combinations thereof) as well as separations performed in solution.Preferably, however, the separations take place in solution.

The term “isoelectric point” or pI, as used in this document, should betaken to mean the pH of the solution in which a protein or otherampholyte has zero mobility in an electric field; hence the pH at whichthe protein or other ampholyte has zero net charge, i.e., no charges oran equal number of positive and negative charges including those due toany extraneous ions bound to the ampholyte molecule. The pH value of theisoelectric point may depend on other ions, except hydrogen andhydroxide ions, present in the solution. Isoelectric point is also knownas “isoelectric pH” (IEP or IpH).

Preferably, the isoelectric focussing in the module as described heretakes place in reduced, or preferably the absence of electroosmoticflow. This may be achieved by use of suitable substrates, as describedin further detail below.

Isoelectric Focussing (IEF) Module The isoelectric focussing modulecomprises a substrate (generally of a planar configuration) which has achannel. The isoelectric focussing module described here is sometimesalso referred to as a “cartridge”, and the isoelectric focussingtechnique and module as “CIEF” (capillary isoelectric focussing).

Channel

The channel is of generally elongate disposition, and preferably linear.The channel may be tubular in construction, but is preferably open alongat least a portion of its length. Preferably, the channel is opensubstantially along the whole of its length, so that it has the shape ofa trough or open channel on the substrate.

The dimensions of the channel are generally in the order of themicrometre range. They are compatible with for example, microcapillarydimensions. By microcapillary or capillary, we refer to a narrow smalldiameter tube, preferably one which is capable of exerting capillaryeffects on a liquid, such as water. It will be appreciated that anycapillary, such as a glass capillary (suitably modified as describedbelow) or a plastic capillary, may be used for the purposes describedhere in place of the channel, provided that it is openable to expose andenable access to the separated components.

Preferably, the channel has a linear dimension, for example, width,depth or diameter of between 1 to 500 micrometres, preferably between 50to 350 micrometres. However, in preferred embodiments, the channel has alinear dimension (preferably a width) of between 100 to 250 micrometres,or between 50 to 350 micrometers. In highly preferred embodiments, thechannel has a linear dimension (preferably a width) of about 127micrometers or about 150 micrometers or about 175 micrometres, mostpreferably about 175 micrometres. Where the channel is open, the depthof the channel is generally greater than its width.

The channel may be engraved or carved out of the substrate, or themodule may be cast with the channel on it using known casting techniqueswith appropriate moulds. The channel may be burned on the substrate, forexample using laser engraving. The channel may be melted, by use of anappropriate tensioned wire, for example a platinum wire which has beenheated preferably by passing an electric current through it).Preferably, the channel is carved out of the substrate, as a groove.Machining techniques as known in the art may be employed for thispurpose. In preferred embodiments, channel is excavated from thesubstrate such that the walls (or at least one wall of) the channel arecomprised of the substrate material.

In highly preferred embodiments, a plurality of channels is disposed onthe substrate. In preferred embodiments, the channel or channels areformed by laser etching, laser ablation, injection moulding or embossingof the substrate.

The phrase “laser etching” is intended to include any surface treatmentof a substrate using laser light to remove material from the surface ofthe substrate. Accordingly, the “laser etching” includes not only laseretching but also laser machining, laser ablation, and the like. The term“laser ablation” is used to refer to a machining process using ahigh-energy photon laser such as an excimer laser to ablate features ina suitable substrate. The excimer laser can be, for example, of theF.sub.2, ArF, KrCl, KrF, or XeCl type.

The term “injection moulding” is used to refer to a process for mouldingplastic or nonplastic ceramic shapes by injecting a measured quantity ofa molten plastic or ceramic substrate into dies (or moulds). In oneembodiment of the present invention, microanalysis devices may beproduced using injection moulding.

The term “embossing” is used to refer to a process for forming polymer,metal or ceramic shapes by bringing an embossing die into contact with apre-existing blank of polymer, metal or ceramic. A controlled force isapplied between the embossing die and the preexisting blank of materialsuch that the pattern and shape determined by the embossing die ispressed into the pre-existing blank of polymer, metal or ceramic. Theterm “hot embossing” is used to refer to a process for forming polymer,metal, or ceramic shapes by bringing an embossing die into contact witha heated pre-existing blank of polymer, metal, or ceramic. Thepre-existing blank of material is heated such that it conforms to theembossing die as a controlled force is applied between the embossing dieand the pre-existing blank. The resulting polymer, metal, or ceramicshape is cooled and then removed from the embossing die.

Open Channel

The isoelectric focussing module comprises means for exposing thechannel along at least a portion of its length. Exposure of the channelin this manner thereby exposes the sample or component(s) therewithin,and allows them to be accessed, preferably for MALDI analysis. In highlypreferred embodiments, the channel is an “open” channel, by which wemean that at least a portion, preferably a substantial portion, of thelength of the channel is not closed or sealed. In other words, in suchpreferred embodiments, the channel adopts the configuration of a trough,being open on one long side. The opening should be at least as wide asnecessary for access to the contents of the channel, for example thesamples, and preferably the separated and focussed components of thesamples, for example, proteins. Preferably, the length of the openingencompasses all or substantially all of the focussed components orproteins.

However, it will be appreciated that closed channels may be used,provided that they are provided with means for opening them. Forexample, closed capillaries may be employed for isoelectric focussing,if they are provided with fracture points to allow them to be splitlengthways. Furthermore, a capillary may be formed by mating two planarmembers each comprising a groove. Isoelectric focussing may then becarried out within the capillary channel, following which the planarmembers may be separated for access to the focussed proteins.

Substrate

The substrate may be formed of any suitable material for isoelectricfocussing, for example, plastics, polymers, ceramic, glass or compositematerials, as known in the art. Generally, any non conducting materialmay be suitable for use as the substrate.

The substrate may be generally elongate, and preferably rectangular inshape. Although any size of the substrate may be employed, the term“substrate” as used here preferably refers to any material that can bemicrofabricated, e.g., dry etched, wet etched, laser etched, moulded orembossed, to have desired miniaturized surface features. In addition,microstructures can be formed on the surface of a substrate by addingmaterial thereto, for example, polymer channels can be formed on thesurface of a glass substrate using photo-imageable polyimide.Preferably, the substrate is capable of being microfabricated in such amanner as to form features in, on and/or through the surface of thesubstrate. Such preferred features include channels as described infurther detail below.

The substrate can be a polymer, a ceramic, a glass, a metal, a compositethereof, a laminate thereof, or the like. By “composite” we mean acomposition comprised of unlike materials. The composite may be a blockcomposite, e.g., an A-B-A block composite, an A-B-C block composite, orthe like. Alternatively, the composite may be a heterogeneous, i.e., inwhich the materials are distinct or in separate phases, or homogeneouscombination of unlike materials. As used herein, the term “composite” isused to include a “laminate” composite. A “laminate” refers to acomposite material formed from several different bonded layers of sameor different materials. Other preferred composite substrates includepolymer laminates, polymer-metal laminates, e.g., polymer coated withcopper, a ceramic-in-metal or a polymer-in-metal composite.

Elements of the device, including but not limited to the platecomprising the channel(s) may be comprised of the substrate.Furthermore, the lid or cover plate where present may also be comprisedof the substrate.

Particularly preferred substrates are those which display lowelectroosmotic flow (EOF). For example, materials whose surface groupsare not substantially charged, for example plastics, are suitable forthis purpose. Materials with charged surface groups may also be used,but are less preferred.

Glass capillary channels, for example, produce strong electro-osmoticflow (EOF) under applied electric field, while most of the plasticsubstrates do not have many ionizable chemical functional groups, andhence, exhibit very weak electro-osmotic flow (EOF) (Soper, S. A., Ford,S. M., Qi, S., McCarley, R. L., Kelly, K., Murphy, M. C., Anal. Chem.2000, 72, 642A-651A). The EOF is an important driving force for movingchemicals inside the microchanel during capillary zone electrophoresis.However, the EOF has to be eliminated in capillary isoelectric focusingas described here for the formation of stable pH gradient by carrierampholyte under the applied electric field (Wehr, T., Rodriguez-Diaz,R., Zhu, M., Capillary Electrophoresis of Proteins, Marcel Dekker, Inc.,New York, 1999). Plastics substrates generally do not have manyionisable chemical functional groups, and they therefore exhibit weakelectroosmotic flow (if any). Plastic substrates are therefore preferredas substrates.

Where materials with charged surface groups are used, for example,glass, surface charges should preferably be reduced by chemicalmodification in order to reduce EOF. Accordingly, glass and othersimilar substrates are preferably surface treated, derivatised or coatedto reduce surface charges. Any material which is used for coatingcapillary channels in CIEF may be used for this purpose, for exampleacrylamide, hydroxypropyl cellulose, methyl cellulose, Teflon andpolyvinyl alcohol.

The term “surface treatment”, including preferably derivatising orcoating, is used to refer to preparation or modification of the surfaceof a substrate that will be in contact with a sample during separation,preferably one or more walls of the channel, whereby the separationcharacteristics of the device are altered or otherwise enhanced.Preferably, the characteristics of the device are enhanced to reduceelectroosmotic flow. Accordingly, “surface treatment” as used hereinincludes: physical surface adsorptions; covalent bonding of selectedmoieties to functional groups on the surface of treated substrates (suchas to amine, hydroxyl or carboxylic acid groups on condensationpolymers); methods of coating surfaces, including dynamic deactivationof treated surfaces (such as by adding surfactants to media), polymergrafting to the surface of treated substrates (such as polystyrene ordivinyl-benzene) and thin-film deposition of materials.

Protocols for coating with various materials are set out below. Foracrylamide coating, the capillary or channel is washed with 0.5M NaOHfor 30 minutes, then with water for 10 minutes. The capillary or channelis then washed with 0.1M HCl for 5 minutes, followed by washing withwater for 30 minutes. A solution of 5 microliter/ml ofgamma-methacryloxypropyltrimethoxysilane in 50:50 volume ofwater:acetone is made up, and the capillary or channel is washed for onehour in this. The capillary or channel is washed with 4% (w/w)acrylamide, 0.04% (v/v) N,N,N,N-tetramethylethylenediamine (TEMED) and0.5 mg/mL ammonium persulphate solution for 30 minutes. Finally, thecapillary or channel is washed with water and then dried by passingnitrogen through or across it.

For hydroxypropyl cellulose, methyl cellulose, or polyvinyl alcoholcoating, any one of these chemicals can be added to the sample toachieve dynamic coating during isoelectric focussing. Alternatively, thecapillary or channel is coated beforehand by washing capillary with 1-5%solution (of the appropriate chemical). The capillary or channel is thenpurged with dry nitrogen. The thin layer of coating is then immobilizedon the capillary by heating it to 140-160 degrees C.

While the above protocols may be conducted on the capillary or channelitself, it will be appreciated that it is possible, and may be moreconvenient, to treat entire substrate with the channel for this purpose.

Where glass substrates are used, and microfabrication techniques forexample as commonly known in the microelectronics industry, may beemployed to engrave or etch the channel on the glass substrate. Polymersubstrates are also amenable to microfabrication technologies, and suchtechnologies are described in detail in Becker, H., Gärtner, C.,Electrophoresis 2002, 21, 12-26. For example, the plastic devices can beproduced from injection moulding, laser ablation, imprinting or hotembossing. Such fabrication techniques allow the device to be replicatedquickly for mass production with inexpensive methods. These allow theuse of single use disposable devices in medical diagnostics andscreenings.

In highly preferred embodiments, the substrate is made ofpoly(methylmethacrylate) (PMMA) or polycarbonate, and at least one wallof the channel comprises this material.

Carrier Ampholyte

Carrier ampholytes are a heterogeneous mixture of synthetic polymersincorporating a variety of both acidic and basic buffering groups.Ampholyte molecules have net charges that depend on the pH of theenvironment and the number and pKs of the particular mixture of acidicand basic groups on the particular molecule. For isoelectric focusing(IEF), carrier ampholytes are introduced into the channel. In theabsence of an electrical field, the carrier ampholytes are randomlydistributed and establish a uniform pH throughout the gel matrix, aboutpH 7 when creating a pH 3-10 gradient.

When an electrical field is applied across the channel, usually throughan acid electrode solution at the anode (+) and a basic electrodesolution at the cathode (−), all carrier ampholytes with a net chargewill start to migrate. Those with a net negative charge and low pI valuemove toward the anode, those with a net positive charge and a high pIvalue move toward the cathode, and those with no net charge (neutral) donot move. The ampholytes with the more extreme pI values can migratecloser to the appropriate electrode solution before they are titrated tothe pH equal to their pI. Thus the pH gradient is established by themobile carrier ampholytes. At equilibrium, the pH at any point in thegel is determined by the average pI of the soluble carrier ampholytes atthat point. At the same time, charged or neutral molecules, such asprotein components of the sample, also move to their pI points, and arefocused.

The carrier ampholytes may be introduced into the channel, and anelectric field applied to create a pH gradient. Alternatively, or inaddition, the carrier ampholytes are mixed into the sample, and thesample containing the carrier ampholytes is introduced into the channel.

Under the influence of the electrical force the pH gradient will beestablished by the carrier ampholytes, and the protein species migrateand focus (concentrate) at their isoelectric points. The focusing effectof the electrical force is counteracted by diffusion which is directlyproportional to the protein concentration gradient in the zone.Eventually, a steady state is established where the electrokinetictransport of protein into the zone is exactly balanced by the diffusionout of the zone.

A large number of carrier ampholyte mixture are available givingdifferent pH gradients. The optimal pH gradient will depend on thepurpose of the experiment. For screening purposes, a broad rangeinterval (pH 3-10 or similar) may be used. A narrow pH range interval isuseful for careful pI determinations or when analyzing proteins withvery similar pI points. Generally, one should not use a narrowergradient than necessary because the shallower gradient will lead tolonger focusing times and more diffuse bands. When choosing pH gradientone should be aware that the interval stated by the manufacturer canonly be an approximation. The exact gradient obtained depends on manyfactors such as choice of electrolyte solutions, gradient medium (PAA oragarose), focusing time etc.

Carrier ampholyte free CIEF has been demonstrated (Huang, T., Wu, X-Z.,Pawliszyn, J., Anal. Chem. 2000, 72, 47584761), and it is possible touse the methods described in Huang and Pawliszyn for the isoelectricfocussing technique described here. Furthermore, it will be appreciatedthat the pH gradient in the channel can also been generated byimmobilizing acidic or basic ampholytic molecules on the open channelsurface. This is described in detail in Rosengren, A., Bjellqvist, B.,Gasparic, V., U.S. Pat. No. 4,130,470, 1978. However, the use of carrierampholytes is preferred.

A carrier ampholyte which may be used for the isoelectric focussingusing the module and apparatus described here is Pharmalyte 3-10, orBioRad 3-10. This may be used typically from 0.8% to 4% or more,preferably about 1%. Carrier ampholytes are described in detail in U.S.Pat. No. 4,131,534.

Glycerol is a common additive for IEF, as it can prevent proteinsprecipitation when proteins concentration increase around their pIpoints. The glycerol is also an infrared (IR) MALDI matrix for proteinionization. The use of glycerol is preferred in the isoelectricfocussing techniques when it is coupled to IR-MALDI-MS.

Mass Spectrometry

(The text in this and the next section describing MALDI-MS and -TOF isadapted from an article in The Scientist 13 [12]: 18, Jun. 7, 1999).

The methods described here typically employ separation using IEF in afirst dimension, and separation by mass in a second dimension. The massseparation is preferably carried out by mass spectrometry. The IEFmodule is preferably coupled to a mass spectrometer for separation anddetection in the second dimension.

Mass spectrometry (MS) systems typically employ components for smashingand ionizing the target molecules by applying energy and for analyzingthe results. Typically, the molecules are ionised by bombardment with anelectron beam, high-energy ions, or a laser. Ionization charges some ofthe sample molecules, which can either remain intact or fragment into avariety of charged and neutral particles. The ions are accelerated by anelectrostatic or magnetic field in the mass analyzer and separated bydeflection or time of flight to the detector. Some mass analyzers candifferentiate between oxygen at 15.999 Da and the similarly sized NH2ion at 16.021 Da. Mass accuracy is generally cited in parts per million(ppm), and many systems claim mass accuracies of ˜100-200 ppm. A reviewof the considerations for designing mass analysers is provided by BruneeJournal of Mass Spectrometry and Ion Processes, 76:125-237).

Two types of ion detectors are typically employed in mass spectrometers:electron multipliers and microchannel plates. Both technologies are wellsuited to ion detection., although electron multipliers (which consistof several layers of charged dynodes) are considered more stable to highion flux.

The first widely available configurations for MS included an electronbeam ionization source, a scanning quadrupole mass filter, and amultidynode ion detector and were suited primarily for analysis ofsmaller molecules. MS first became useful for protein research when fastatom bombardment (FAB) ionization sources were designed to smash largermolecules (including proteins and peptides up to ˜10 kDa) intomanageable pieces. FAB uses a high-energy (5-10 keV) stream of inert gasparticles to “ballistically ionize” the sample. It is limited by arelatively poor efficiency of target ionization and can lead to highbackgrounds when the ionizing particles themselves break up, ionize, andimpact the detector.

Electron spray ionization (ESI) increased the protein mass range to ˜100k Da. Quadrupole and magnetic sector ESI MS became very valuable tools.ESI uses a high electric field to aerosolize a solution of the targetanalyte; the droplets subdivide until they contain a single analytemolecule that carries a residual charge. Often, ESI-productions carrymultiple charges, which can be a benefit or a problem, depending on yourinstrument and application. Neither FAB nor ESI is suited to workingwith samples in bulk form or on a solid support.

For proteins, ESI MS has in many ways been superseded by MALDI as thehammer and by time-of-flight mass analyzer tubes as the detector. Themethods and apparatus described here preferably employs a MALDI massspectrometer for separation and detection in the second dimension.

Matrix-Assisted Laser Desorption/Ionization (MALDI)

Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)mass spectrometry is a tool for large-molecule analyses, especially forproteins. MALDI-TOF is capable of distinguishing protein and nucleicacid sequence, structure, purity, heterogeneity, cleavage,posttranslational modification, and other molecular characteristics thatare often difficult to study by other means. MALDI is described indetail in Chapman, J. R, Mass Spectrometry of Proteins and Peptides,2001, Humana Press, Dass, C., Principles and practice of biological massspectrometry, 2001, John Wiley & Sons, James, P., Proteome research:mass spectrometry, 2001, Springer, Kellner, R, F. Lottspeich, and H. E.Meyer, Microcharacterization of Proteins, 2nd Ed, 1999, Wiley-VCH,Kinter, M., and N. E. Sherman, Protein Sequencing and IdentificationUsing Tandem Mass Spectrometry, 2000, Wiley Interscience and Siuzdak,G., Mass Spectrometry for Biotechnology, 1996, Academic Press.

MALDI uses pulses of laser light to desorb the analyte from a solidphase directly to an ionized gaseous state. Pulsed lasers had been usedto ionize proteins prior to 1988, but the technique was limited due toprotein light absorption. A metal powder matrix for laser desorption andionization of analytes was first presented in 1987 by Koichi Tanaka andcolleagues (K. Tanaka et al., Shimadzu Corp., Kyoto, Japan, “Proceedingsof the 2nd Japan-China Joint Symposium on Mass Spectrometry,” 185,1987).

The more common MALDI method using an organic photoactive compound waspublished in 1988 by Michael Karas and Franz Hillenkamp (M. Karas, F.Hillenkamp, “Laser desorption of proteins with molecular massesexceeding 10,000 Daltons,” Analytical Chemistry, 60:2299, 1988) and hasbeen more recently reviewed by Ronald Beavis and Brian Chait (R. C.Beavis, B. Chait, “Matrix assisted laser desorption ionizationmass-spectrometry of proteins,” Methods in Enzymology, 270:519, 1996).

In MALDI, the protein is embedded in a medium or matrix bycocrystllization with a photoactive compound such as gentisic acid,4-HCCA (alpha-cyano-4-hydroxycinnamic acid), or dithranol. The typicalmatrix for use with ultraviolet lasers is an aromatic acid with achromophore that strongly absorbs the laser wavelength. Other laserwavelengths are possible, in particular the mid-infrared range where thematrix can be energized by vibrational excitation; different matrixcompounds must be used in this case. The MALDI matrix must meet a numberof requirements simultaneously: be able to embed an isolate analytes(e.g., by co-crystallization), be soluble in solvents compatible withanalyte, be vacuum stable, be able to absorb the laser wavelength, causeco-desorption of the analyte upon laser irradiation and promote analyteionization.

The matrix compound absorbs the light and uses the energy to eject andionize the embedded protein molecules. As the protein does not fragmentduring desorption, MALDI is often referred to as being a “soft”ionization technique. The list of suitable matrix compounds for MALDI isextensive, and include Cyano-4-hydroxyciminamic acid (CHCA),2,5-Dihydroxy benzoic acid (DHB), Alpha CCA, Sinapinic Acid (SA),3-hydroxypicolinic acid (HPA), IAA (Na+), 2-(4-Hydroxyphenylazo)benzoicacid HABA (Na⁺), Dithranol (Na⁺), Retinoic Acid (Na⁺), Succinic acid,2,6-Dihydroxyacetophenone, Ferulic Acid, Caffeic acid, Glycerol and4-Nitroaniline. Preferably, the matrix is added to the dried sampleafter isoelectric focussing. Alternatively, or in addition, the matrixmay be added to the sample such that it is present during theisoelectric focussing.

Although other options are available, most MALDI techniques typicallyilluminate at about 20 mJ cm⁻² using nitrogen lasers (337 nm) orQ-switched neodymium:yttrium-aluminum-garnet (Nd—YAG) lasers withfrequency tripled to 355 nm or quadrupled to 266 nm. Longer wavelengthsare favored for protein work because they are less readily absorbed.

Magnetic sector and quadrupole mass spectrometers work by accelerating astream of ionized sample along a vacuum tube toward an electrostatic ormagnetic field that deflects or filters particles based on momentum ormass-to-charge ratio (m/z). A good review of MS detectors can be foundin Brunee (1987, International Journal of Mass Spectrometry and IonProcesses, 76:125-237).

In time of flight mass spectrometry (TOF MS), the ionized analytemolecules and fragments are accelerated in an electrostatic field to acommon kinetic energy. If all the ions have the same initial kineticenergy, lighter ions travel faster and heavier ions with the samemomentum travel more slowly. The ionized particles enter at one end ofthe time-of-flight tube, which typically comprises a long, empty tubefor free flight, and the number of ions reaching a detector at the otherend is recorded in a time-dependent manner. Assuming all the ions havethe same electrical charge, the lightest ions reach the detector firstand the heaviest arrive last The entire mass spectrum is typicallyrecorded in a fraction of a second as ion flux versus time.

For TOF to work, the time at which the ions leave the source must beprecisely controlled and defined. While MALDI ionization techniques havebeen coupled with quadrupole ion and magnetic sector mass analyzers, thecommonest modern combination is with time-of-flight tubes, because theionization event automatically provides the start pulse for the clock.The short duration of laser pulsing makes MALDI a particularly suitablematch for TOF MS. Typically, flight-tube lengths are a couple of metersand flight times are ˜100 ms-thousands of times longer than thenanosecond laser pulses.

The mass range of a TOF instrument is generally limited by the detectortechnology employed. The high m/z ions end up travelling very slowly andare very poorly detected by conventional detectors. Instruments such asGSG Analytical Instruments' Future MALDI-TOF spectrometer extend themass range of MALDI-TOF out to 1,000,000 Da with the help of a two-stagedetector that captures the high m/z particles more effectively and afast (1 GHz) digitizer to increase resolution. Accordingly, suchinstruments are preferred for use in detecting high molecular weightentities in the methods and apparatus described here.

The simplest TOF instruments have a linear configuration, with thedetector placed at the end of the flight tube; this is a typicalconfiguration in MALDI-TOF instruments which are currently available.

During sample desorption and ionization, analyte particles can leave thesurface of the protein-matrix cocrystal with a small but variable amountof kinetic energy in addition to the energy imparted by the accelerationprocess. This variable kinetic energy has the effect of “smearing” themass-to-charge ratio of a specific analyte fragment over a small timerange, decreasing the signal-to-noise ratio and broadening the analytebands, but it can be largely eliminated in a couple of ways. The firstis time lag focusing or delayed extraction, in which newly formed ionsare held close to the surface of the protein-matrix cocrystal with a lowvoltage (generally 1 keV or so) pulse before applying the mainacceleration pulse (generally 20-30 keV). Most instruments nowincorporate this feature. Time lag focusing or delayed extraction isdescribed in further detail in W. C. Wiley, I. H. McLaren, Review ofScientific Instruments, 26:1150-7, 1955 and B. Spengler, R. J. Cotter,“Ultraviolet laser desorption/ionization mass spectrometry of proteinsabove 100,000 Daltons by pulsed ion extraction time of flight analysis,”Analytical Chemistry, 62:793-6, 1990.

The second way to focus an ion band is to change the TOF geometry byadding a reflectron to the end of the flight tube and moving thedetector(s). A reflectron or “ion mirror” consists of a series ofelectrostatic and magnetic fields that collect and redirect the ions ina controlled manner. Ions with a given m/z slow down as they approachthe reflectron mirror, focus into a tighter packet, and are thenrepelled either at an angle toward a detector at the end of a secondstage of flight tube or backward along the same tube to a detectorplaced near the ion source. For many applications, reflectron-based TOFtubes give sharper signals by reducing the effects of initial kineticenergy differences.

Because reflections effectively increase—almost double—the TOFfree-flight path, they increase resolution and therefore improve massaccuracy. Reflectron technology also allows researchers to studymolecular structure of ions via postsource decay, in which ionizedfragments decompose further in the flight tube and the secondaryproducts provide additional information about the structure of theoriginal ion. The information gained from postsource decay detection issimilar to that provided by tandem MS (MS/MS), where ions areintentionally refragmented after passage through a mass analyzer and thesecondary fragmentation products are examined in a second mass analyzer.

Examples of reflectron-based MALDI-TOF instruments include Comstock'sRTOF-260 instrument, which is a reflectron-based version of itsLTOF-160. PerSeptive Biosystems (a division of PE Biosystems) offers theVoyager DE™ workstation, 4700 TOF/TOF and the Voyager DE-PRO.

Micromass and Kore produce the TofSpec-2E and R-500 TOF MS,respectively. The M@LDI, made by Micromass, may also be used.

The several reflectron systems offered by Bruker Daltonics, includingthe customizable REFLEX m system and the BIFLEX III system for high-endresearch, the Autoflex and the Ultraflex, may also be used.

Reflectron-based instruments, such as the Kompact DISCOVERY and theKompact SEQ, made by Kratos Analytical, a Shimadzu company, may also beused Kratos' reflectrons have a design incorporating a curved fieldrather than stepped or tiered linear fields. In normal ion reflectionconfigurations, many of the postsource decay ions are “out oftime-focus” and are therefore lost. Most instruments collect only about10 percent of the range of postsource decay particles, necessitatingrepeated experiments at different collection points. The curved fieldallows collection of the entire range of postsource decay products fromone laser pulse without rastering or scanning and eliminates the need tocompile data from sequential experiments. Shimadzu also produces theAXIMA-CFR-plus and AXIMA-QIT.

Multisample target formats are becoming more important to users, andmany companies have started to offer them. For example, BioMolecularInstruments, a division of Thermo BioAnalysis, recently introduced theDynamo, which is highly automated and incorporates a video camera in theionization chamber for direct sample monitoring. The Bruker REFLEX IIIand BIFLEX III instruments both offer integration with Bruker's SCOUT384 automated sampler. The SCOUT 384 uses standard microtiter plateformats and an X-Y positioner with 4 mm accuracy for unattended dataacquisition from up to 1,536 samples.

It will be appreciated that other MALDI mass spectrometers, other thanMALDI-TOF spectrometers, may be used. For example, FTMS (FourierTransform Mass Spectrometers) may also be used or combined with themodule as described here.

Specific Embodiments

Preferred embodiments of the present invention will now be describedwith reference to the accompanying Figures, wherein like numerals referto like elements throughout. The terminology used in the descriptionpresented herein is intended to be interpreted in its broadestreasonable manner, even though it is being utilized in conjunction witha detailed description of certain specific preferred embodiments of thepresent invention. This is further emphasized below with respect to someparticular terms used herein. Any terminology intended to be interpretedby the reader in any restricted manner will be overtly and specificallydefined as such in this specification.

FIGS. 1A and 1B show a first embodiment of the isoelectric focussingmodule with a single microchannel. Module comprises a substrate 1 ofgenerally planar configuration, made of poly(methylmethacrylate) (PMMA)or polycarbonate. The substrate 1 comprises a piece of a PMMA platehaving dimensions of 90 mm×30 mm×3 mm. A channel 2, which in thisembodiment is an open channel, is carved, built or etched out of thesubstrate. Reservoirs 3 and 4 machined from the substrate and arepositioned at opposite sides of the channel and carry electrolyte(anolyte and catholyte). The reservoirs 3, 4 are separated from the openchannel by agarose plugs 31. The agarose plugs are set in the boundaryof the reservoir and the channel, and allow electrical conductivity tobe maintained between the electrolyte solution and the contents of thechannel (typically a sample to be separated, see below). Mixing betweenthe contents of the channel and the reservoir is prevented, however, bythe presence of the agarose plugs. Mixing may also be prevented by theuse of a gel plug, such as an acrylamide gel plug or an agar gel plug,or any other suitable gel plug which prevents mixing but conductselectricity. Mixing may be prevented by increasing the viscosity of thesample, by for example, adding glycerol to it The viscosity of theelectrolytes, or one or both of the anolyte and catholyte, may beincreased alternatively, or in addition to increasing the viscosity ofthe sample. For example, methylcellulose may be added to the or eachelectrolyte(s).

It will be understood that the presence of the agarose or gel plug isoptional, provided that mixing is minimised. For example, the channelmay have a narrower profile at the region where it joins the reservoir;the narrow portion will substantially reduce convection and thereforeany mixing between the components of the channel and the reservoir.

FIGS. 4A to 4C show a second embodiment of the isoelectric focussingmodule, which contains multiple channels 2. Each of the channels in thisembodiment may be fabricated as described above for the single channelembodiment. In a preferred embodiment, the channels are parallel witheach other, or substantially so. The channels may each have individualreservoirs 3, 4 connected to them, or preferably may be joined at eachend to a common reservoir 3, 4 shared by all the channels. As describedabove, each channel may preferably have an agarose gel or plug,preferably at each end, to reduce mixing with the electrolyte contentsof the reservoir.

The channel or microchannel 2 can have a variety of shapes or profiles;indeed, any profile which is conducive to isoelectric focussing and openat one edge may be used. Examples of individual channel profiles areshown in FIGS. 2A to 2D. Thus, the channel 2 may have a flat bottom andstraight walls, so that it adopts a flat U shape (FIG. 2A). The channel2 may have a curved or bowed or convex profile, and straight walls, thushaving a typical “U” shape (FIG. 2B. The channel 2 may have curved wallsand a straight base (FIG. 2C) or substantially curved walls adopting acurved “V” shape (FIG. 2D). A variant of the profile of FIG. 2D, withstraight walls, i.e., a straight “V” shape, may be employed. In each ofthese cases, the channel may be carved, etched, or gouged out of thesubstrate 1. FIG. 4B shows a profile of the module in a secondembodiment of the device, showing the multiple channels 2 on thesubstrate 1.

In the first and second embodiments described above, the reservoirs 3, 4are provided on the same piece of substrate as the channel, i.e., theyare provided “in cis”.

In other embodiments of the module or device, however, the reservoirsare not located on the same substrate as the channel. Rather, they areprovided on a separate lid or cover plate 7, as illustrated for a thirdembodiment in FIGS. 5A to 5C. As can be seen from FIGS. 5B and 5C, aseparate cover plate or lid 7 is provided to hold the reservoirs 3, 4.The reservoirs 3 and 4 may be provided as tubular compartments, whichare capable of holding electrolyte. They may for example have a conicalshape, or a cylinder having a conical end. In the embodiment shown inFIGS. 5A to 5C, the electrolyte reservoirs are modified from amicropipette with a sharp tip of about 10 μm. They are attached to thecover plate as shown in FIGS. 5C, 6A and 6B. In such embodiments, thereservoirs 3, 4 can be said to be provided “in trans”.

The tips of the micropipettes are filled with a thin layer of agarosegel to prevent mixing of the electrolytes and the sample while allowingions to migrate through the junction It will be seen from FIGS. 5C, 6Aand 6B that distal portions of the reservoirs 3, 4 extend across the lid7, and mate with respective ends of the channels 2 when the lid isplaced on the substrate 1. For this purpose, suitably sized andpositioned holes may be drilled on the lid 7 to hold the reservoirs 3, 4in position.

The lid or cover plate 7 and the base 1 may further comprise guidingmeans 71, as shown in FIGS. 5A, 5B, 7A and 7B for guiding the lid 7 tooverlay the base or substrate 1. The guiding means may comprise markingson the lid 7 and substrate 1, or preferably physical guiding means suchas a peg and hole arrangement, a tongue and groove arrangement, etc.Preferably, the lid 7 comprises a hole 71 and the base or substrate 1comprises a peg or post 71 (or vice versa). The guiding means enablesprecise alignment between the base and the lid, so that the channels andrecesses are matched to their correct positions, and the outlets of theelectrolyte reservoirs are pointed to the channels. Preferably theguiding means 71 is asymmetrically placed on the lid and base, so thatthe two can only be mated in one orientation.

The cover plate 7 preferably is not completely flat, particular atpoints abutting the channel or channels. This is because a completelyflat cover plate would cause the sample in the microchannel 2 to contactthe cover plate 7 and thereby diffuse out of the channel to the gapbetween two plates by capillary action. Accordingly, in a preferredembodiment, the lid or cover plate 7 comprises one or more grooves orrecesses 8, preferably the same number of recesses as there arechannels, on one face (i.e., the face which abuts the microchannels whenthe lid is in place). The or each groove or recess is positioned suchthat when the lid is mated with the first planar member, no substantialleakage of sample contained in the channel occurs. Preferably, thelength and width of the recess or recesses are at least as great as achannel or respective channels in the first planar member. The groovesmay be machined on the corresponding opposite side of the microchannelon the cover plate to prevent the sample from diffusing out to the gap.The arrangement of recesses 8 on the cover plate 7 is shown in FIGS. 6A(lid open) and 6B (lid closed).

FIGS. 7A to 7C show a fourth embodiment of the isoelectric focussingmodule, which is identical with the third embodiment except that itcontains multiple channels 2. Each of the channels in this embodimentmay be fabricated as described above for the single channel embodimentIn a preferred embodiment, the channels are parallel with each other, orsubstantially so. The reservoirs 3, 4, serving the channels are locatedon a separate lid or cover plate 7, as described above. Each reservoirmay preferably have an agarose gel or plug, to prevent or reduce mixingbetween the electrolyte contents of the reservoir, and the contents ofthe channels. A profile showing the arrangement of the multiplereservoirs 3, 4 on the lid 7, and the recesses or grooves 8, togetherwith the substrate 1 comprising multiple channels 2, is shown in FIG. 8A(lid open) and FIG. 8B (lid closed).

The use of a lid in the third and fourth embodiments is advantageous inthat it reduces evaporation of the sample in the channel or channels.Evaporation is a particular problem because of the small volume andlarge surface area of the sample in the channel. The lid maintains ahumid atmosphere above the sample, and prevents drying out. Furthermore,the presence of a cover also prevents carbon dioxide from the airdissolving into the sample, and perturbing the pH gradient established.

It will be appreciated, however, that the presence of a cover is notstrictly necessary, and other means may be used to control drying and pHperturbation. For example, isoelectric focussing with embodiments oneand two with open channels may be carried out in a controlledatmosphere, particularly one with humidity conducive to non-evaporation(i.e., one high in humidity—high relative humidity). Therefore, ahumidity controlled chamber may be used. Furthermore, the controlledatmosphere may be depleted of carbon dioxide. For this purpose, a simpleair-tight chamber may be used; the chamber may contain a carbon dioxidedepleting agent, such as an alkali metal hydroxide (NaOH, KOH) or analkaline metal hydroxide (Ca(OH)₂), or other chemicals such as NaCO₃,etc. The chamber may comprise a mister, or simply a source of water, formaintaining high humidity.

Furthermore, or alternatively, evaporation may be reduced by reducingthe vapour pressure of the solvent in the sample, for example bycooling. For this purpose, the temperature of the module or thesubstrate, or its surroundings may be reduced. For this purpose, thecartridge or module is mounted on an aluminium block which in turn wasimmersed in an ice bath. The temperature can also be controlled byattaching the cartridge or module onto a thermoelectric cooler.

For isoelectric focussing in the module, anolyte and catholyte areintroduced into the reservoirs. 100 mM potassium hydroxide in 1.5%methylcellulose is used as catholyte and 50 mM phosphoric acid in 1.5%methylcellulose is used as anolyte. A sample—for example a samplecontaining proteins such as a cell extract—is introduced into thechannel. As the module and device are particularly useful for separationand analysis of proteins in cell, tissue, or organ samples, thefollowing description will be based on separation of such proteins incellular samples.

The sample may contain a carrier ampholyte as known in the art, and asdescribed above. A voltage of from about 500V to 5 kV is then appliedacross the channel 2 by means of electrodes introduced into thereservoirs 3 and 4. For this purpose, the module or apparatus maycomprise a power supply (not shown), which is capable of generating anelectrical potential between two points, preferably the electrodes 3, 4.The power supply is preferably a DC power supply, as known in the artfor use in electrophoresis devices. Examples include, but are notlimited to, PowerPac Basic power supply, PowerPac 3000 power supply,PowerPac 1000 power supply, PowerPac 200 power supply, and PowerPac 300power supply, produced by BioRad. Other suitable power supplies includethe EC105 Power Supply, EC135-90 Power Supply, EC250-90 Power Supply,EC4000P Programmable High Voltage Power Supply, EC570-90 Power Supply,EC600-90 High Voltage Power Supply, EC6000-90 High Voltage Power Supply,EC PRO6000 Power Supply, EC1000-90 Power Supply, made by Thermo EC(Thermo Savant/Thermo EC Holbrook, N.Y., United States).

The power supply may be linked to the electrodes 3, 4 by means of wires.The power supply may further comprise control means, by which anoperator is able to control various parameters. For example, the controlmeans may allow the operator to vary the potential difference (voltage).The control means may enable the current to be modified, for example,the current across the channel. Control of the voltage and current isadvantageous because it enables the amount of Joule heating(voltage×current) to be adjusted.

Application of the voltage across the channel causes a pH gradient todevelop along it, as described in detail above. The molecules, proteinsor other components of the sample then migrate along the channel and arefocussed at points according to their respective pI points. Isoelectricseparation and focussing of the components therefore takes place alongthe channel.

The voltage is applied for a suitable amount of time to allow focussing,for example, about 5 minutes. Protein focussing into a narrow zone isindicated by a drop in the focusing current to a constant value.Following this, the voltage applied across the capillary channel may begradually increased to tighten the focusing zones. The use of modulescomprising multiple channels (e.g., embodiments two and four describedabove) is advantageous, as many different samples may be processed atthe same time. Migration and focussing of the proteins may also bemonitored by means of suitable stains.

After the proteins are separated, the sample is dried to retain theseparated components of the sample, for example proteins, at the pointsat which they are focussed. Any suitable means for removing the solventin the sample may be used, for example, application of a stream of warmair, preferably dry air over the module. Lyophilisation, vacuum dryingor freeze drying, may also be employed for this purpose. Application ofan electrical potential across the channel, which causes Joule heating,may be employed to evaporate the solvent. Proteins “frozen” in positionare accessible because of the open nature of the channel, and may thenbe analysed by any suitable means. In preferred embodiments, a massspectrometry technique is used to analyse the proteins, for exampleMALDI-TOF. For this purpose, and as noted above, the MALDI matrix may beadded to the sample. Alternatively, it may be applied to the driedfocussed proteins in the channel.

The isoelectric focussing module may or may not then be coated with anelectrical conducting thin film or layer by different means beforeperforming MALDI-MS. The conductive coating may be achieved by vacuumdeposition of metallic or conductive layer, or by painting a layer ofconductive material, or by use of conductive adhesive tape, or by othermethods. In the preferred embodiment, the isoelectric focussing moduleis not coated with any conductive coating.

The isoelectric focussing module (including substrate comprising thechannels) is then mounted on a standard MALDI plate for the MALDIprocedures. The cartridge may therefore be loaded into a translationalstage, for example an X-Y translational stage, in the MALDI ionizationsource. The isoelectric focussing module may be mounted directly on theMALDI plate, or the isoelectric focussing microchannel may be fabricateddirectly on the MALDI plate, or an adapter may be fabricated to hold theisoelectric focussing module onto the MALDI plate. FIG. 3 (and also FIG.4C) shows a cross section of a module comprising the planar substrateand channel, which is mounted on a stage or adapter 5. The adaptor 5 isalso depicted in FIG. 10. The MALDI laser 6 is then focussed on thecentre of the channel or microchannel, and the MALDI plate is movedslowly across the laser 6, while maintaining the laser beam 6 on thecentre of the channel. The translational stage may be moved toconsecutively bring the whole open channel to the focused laser spot.The MALDI laser 6 ionizes the proteins, and analysed using time offlight (TOF) or other means as described in detail above. The proteinmolecular ions from the MALDI source may be fragmented by post sourcedecay or collisionally activated dissociation for proteinidentification.

In highly preferred embodiments, the separation of protein samples inopen channels is preferably done in parallel format where multiplemicrochannels are built on a cartridge. Such embodiments are the secondand fourth embodiments, illustrated in FIGS. 4A-C and 7A-C.

The parallel CIEF separation can take less than 5 minutes. The detectionof protein in the second dimension requires scanning the channel overthe focused laser in the MS ionization source region. How fast thechannel for MALDI ionization may be moved depends on the repetition rateof the desorption/ionization laser. Nitrogen lasers commonly used inMALDI normally operate at 10-20 Hz. However, diode-pump solid statelasers can operate in several kHz repetition rates, and are thereforepreferred. Therefore, in such preferred embodiments, a complete scan ofthe whole channel using such lasers can be completed within minutes.High throughput, high sensitive protein separation and characterizationthrough their pI points and molecular weights can be achieved using ourapparatus with minimal sample consumption for clinical application.

The protein ionization process can be significantly suppressed byimpurity and salts. Separation of the sample components by open channelCIEF prior to the MALDI-MS analysis minimizes the potential of signalsuppression due to the presence of other sample components in same spot.The effect of salts (cations and anions) in the sample can be minimizedbecause ions will migrate out of the separation channel under theinfluence of the applied electric field. The focused laser into smallspot provides high spatial resolution allowing analysis of sample only afew micro meter size. Therefore, the high resolution separation ofproteins by capillary isoelectric focusing can be retained in the seconddimension by laser desorption/ionization. Open channel CIEF-MALDI asdescribed in this document is expected to have high sensitivity becausethe focused proteins are concentrated on a small spot in a narrowchannel.

The invention is described further, for the purpose of illustrationonly, in the following examples.

EXAMPLES Example 1 Materials and Reagents

Poly(methylmethacrylate) (PMMA) is purchased from a local supplier(Swees Engineering Co. (PTE) Ltd.). Myoglobin, glycerol, pharmalyte,methylcellulose are ordered from Sigma Chemicals. All other chemicalsare acquired from Aldrich Chemicals. All solutions are prepared usingwater purified by a Nanopure water system. Platinum wires are suppliedby Fine Metal Crop.

Example 2 Fabrication of Open Microchannel

Pieces of PMMA plates, 90 mm by 30 mm and 3 mm thickness, are cut from araw plastics plate. Platinum wires with diameters of 0.005 inches and0.007 inches are used to imprint the channels in the plastic substrate.A platinum wire about 150 mm in length is stretched taut by clamping theends to a wire tension bow. The PMMA and a glass plate sandwich theplatinum wire and are clamped together between two aluminium blocks.Electrical current is passed through the platinum wire until it is redhot while pressure on the aluminium block is applied by tightening theclamp. When the assembly is cooled down completely, the clamp isreleased and the platinum wire is pulled away from the plastic to revealthe channel.

In one design, holes at the ends of the channel are drilled for theelectrolyte reservoirs. In another design, a cover plate is used tocover up the open microchannel during the focusing to minimize isevaporation and carbon dioxide absorption to sample solution. The coverplate was fabricated from PMMA using standard machining methods.

Example 3 Open Channel Capillary Isoelectric Focusing

Myoglobin is used as model protein for the method development because ithas a brownish colour and can be detected by human eyes. The sample isprepared in the concentration of 0.02 μg/μl of myoglobin, 1% ofPharmalyte 3-10. About 5 μl of sample is applied to the openmicrochannel by a micropipette. The sample spreads out evenly in themicrochannel by capillary action.

100 mM potassium hydroxide in 1.5% methylcellulose is used as catholyteand 50 mM phosphoric acid in 1.5% methylcellulose is used as anolyte.The methylcellulose significantly increases the viscosity of theelectrolytes. Two different setups are used for the isoelectric focusingas shown in FIGS. 1 to 4 and in FIGS. 5 to 8.

In the first design (see for example FIG. 4), the electrolytes arefilled in two reservoirs at the ends of the open microchannelrespectively. The sample in the microchannel and the electrolytes in thereservoirs are separated by an agarose gel set in the boundary ofreservoir and microchannel. The high viscosity of the electrolytes andthe agarose gel prevents mixing of the sample with the electrolytesduring the focusing while still allowing ion passage through the agarosegel. Platinum wires are attached to the reservoirs for electricalcontact.

In the second design (see for example FIG. 7), the microchannel iscovered up by a cover plate, and the electrolyte reservoirs are madefrom modified micropipettes. Two holes are drilled in the cover plate toreceive the micropipettes, and the micropipettes are attached to thecover plate as shown in FIGS. 6A and 6B.

The tip of the micropipette is about 10 μm in diameter and is pointeddownward to the microchannel. The tips of the micropipettes are filledwith a thin layer of agarose gel to prevent mixing of the electrolytesand the sample while allowing ions to migrate through the junction. Thereservoirs modified from the micropipettes are filled with the highviscosity catholyte and anolyte. Electrical contact is through platinumwires immersed in the electrolyte solutions.

The electrical voltage is varied from 500 V initially to 5 kV in thefinal stage of focusing depending on the electrical current passingthrough the channel. The progress of the focusing is monitored byrecording picture using a Nikon D-100 digital camera.

FIG. 9 shows the progress of IEF of myoglobin in a PMMA open channelwith 0.007 inches width (175 micrometres). The pictures are recordedusing a digital camera After proteins are focused into a narrow zone, asindicated by a drop in the focusing current to a constant value, thevoltage applied across the capillary channel is gradually increased totighten the focusing zones. In addition, Joule heating is increased toevaporate the solvent in the microchannel so that the focused driedprotein does not move when the applied voltage is terminated.

It is more convenient to carry out the focusing in “open system”, wherethe cartridges are essentially open to the atmosphere. However, in someexperiments, we found it advantageous to control the atmospheresurrounding the cartridge, in particular, the humidity and carbondioxide concentration. Without a controlled environment, there is apossibility that the sample would dry out. Furthermore, there is apossibility that carbon dioxide in air would slowly dissolve into thesample solution, perturbing the pH gradient and possibly decreasing theresolution of the separation. If the sample was left in air for a longtime, the amount of carbon dioxide dissolving into the sample might behigh enough to completely destroy the pH gradient. Use of suitablebuffers which minimise pH effects from external sources can also beemployed in addition to use of a humidity and CO₂ controlledenvironment.

In these experiments, we placed the PMMA cartridge with the opencapillary charnel in a controlled environment to produce a “closedsystem”. Such controlled environments allow the amount of humidity(relative humidity) and carbon dioxide to be specifically controlled.Although separation was satisfactory in the “open” configuration, wefound better results using controlled humidity and carbon dioxide freeatmosphere.

Example 4 Coupling MALDI-MS

MALDI-MS experiments are performed in Bruker Daltonics Autoflex MALDItime-of-flight mass spectrometer (TOF-MS) operating in the linear mode.

An adapter is fabricated to hold the plastic CIEF cartridge on thestandard Bruker MALDI sample plate as shown in FIGS. 3, 4C, 5C, 7C and10. Saturated sinapinic acid in acetonitrile with 1% acetic acid isloaded on top of the dried sample in the microchannel. The matrix isadded slowly in several small amounts to the microchannel to preventdegradation and broadening of the focused zone.

The solvent is then allowed to evaporate by exposing to air at roomtemperature. As acetonitrile has a very low vapour pressure, exposure toair enables the solvent to evaporate.

After solvent evaporation, the cartridge is put into an adapter on thestandard Bruker Daltonics MALDI plate (FIG. 10). The MALDI with delayedion extraction is carried out with a conventional nitrogen laseroperating at 337 nm wavelength. The MALDI laser is focused on the centreof the microchannel and the MALDI plate is moved slowly across the laserwhile maintaining the laser on the centre of the microchannel.

As shown in FIG. 11, the myoglobin signal from the focused zone in PMMAchannel has comparable resolution and sensitivity to sample directlyapplied to standard stainless steel MALDI plate. The carrier ampholytesdid not affect the ionization of myoglobin.

Alternatively, a small amount of glycerol (1-2%) may be mixed with thesample before loading into the microchannel. Glycerol can preventproteins precipitating when protein concentrations increase around theirpI points. After isoelectric focusing, the solvent may be evaporated byeither freeze dry or by increasing the applied voltage for Jouleheating. The low vapour pressure glycerol, carrier ampholyte andproteins will stay in the channel, the proteins can then be ionized byapplying an IR laser for IR MALDI-MS because glycerol is a good IR MALDImatrix (Siegel, M. M., Tabei, K, Kunz, A., Hollander, I. J., Hamann, P.R, Bell, D. H., Berkenkamp, S., Hillenkamp, F., Anal Chem. 1997, 69,2716-2726).

Example 5 Separation of Liver Proteins

Proteins are extracted from pig liver using the following protocol.Porcine liver is mixed with 2× volume of deionized water and homogenizedusing a blender. The cells are lysed using a sonicator. The mixture iscentrifuged, and the supernatant is diluted 2 times with deionized waterand used for open channel CIEF without further purification. Openchannel CIEF is carried out essentially as described in Example 3, andMALDI separation may be carried out essentially as described in Example4.

The results of the open channel CIEF of pig liver proteins are shown inFIGS. 12A and 12B. FIG. 12B shows a magnified portion of FIG. 12A. Threebrown spots representing separated proteins are observed at aroundposition 8 of the ruler, indicated by arrows. The ruler is position toprovide an estimate of the pI in the particular corresponding region ofthe channel; accordingly, it will be seen that the method andmodule/apparatus described here is capable of resolving three visibleproteins having similar or close pI values from a complex liver extract.

The three spots represent only three of the proteins present in theliver extract, which are visible to the eye. Needless to say, the liverextract comprises many other proteins, which have been separated by theisoelectric focussing module and method described here.

Other Aspects

FIG. 4A illustrates a top view of one embodiment of the invention. Aplurality of open micro-capillary channels (2) are etched or built on asubstrate (1). Two electrolyte reservoirs (3 and 4) are etched or builton the sample substrate. One reservoir is on one end of themicro-capillary channels, another is on the other end of themicro-capillary channels. The two ends of the open capillary channelsare connected to two reservoirs respectively through narrow channels,semi-permeable membrane or gel in the boundary to prevent mixing ofsample with anolyte or catholyte. The reservoirs are for anolyte andcatholyte solutions. The substrate can be any non-conductive materialssuch as polymeric, ceramic, glass, or composite materials. FIG. 4B showsthe cross-section of the multiple open micro-capillary channels (2). Thecross-section of the channels is not restricted to a rectangular shapebut it can be any shape. Each of the different protein samples will beloaded into one unique channel. When an electric potential is appliedacross the anolyte and catholyte, the proteins will move under theelectric field to their pI points. The separated proteins can bedirectly ionized with a MALDI laser on top of the open channels withoutmobilizing the proteins. Instead of mobilizing the separated proteins,the whole CIEF cartridge will be put on a translational stage and theproteins will be brought to the MALDI laser spot by moving thetranslational stage. As a result, the high resolution achievable withCIEF is preserved in the detection process. Moreover, many opencapillary channels can be built on a CIEF cartridge, the number ofchannels is not fixed rather it can be as many as the cartridge canhold. Therefore, many samples can be separated in parallel for highthroughput application.

The CIEF separation can be done with or without carrier ampholyte. Inone embodiment, pH gradient can be formed by immobilizing some acidicand basic compounds on the capillary wall. MALDI matrix, such asglycerol (0-50%), will be added to the protein samples before CIEFseparation. The glycerol, in this example, will serve as infrared MALDImatrix for the protein ionization, it also prevents precipitation ofprotein in the focused zone and minimizes electroosmotic flow. AfterCIEF separation, the CIEF cartridge is quickly cooled down to freeze thesample to prevent movement of the separated zones. Then, the cartridgeis put into a vacuum chamber to freeze dry the sample, i.e. to evaporatethe ice. What remains in the MOC-CIEF cartridge is the low vapourpressure compounds: mainly separated proteins and MALDI matrix such asglycerol. The cartridge is then loaded into the translational stage inthe MALDI ionization source. FIG. 4C shows the cross section of themultiple open channels (2) capillary isoelectric focusing cartridge (1)mounted on a XY-translational stage (5) in MALDI source. A MALDI laser(6) will be focused on one spot of the open capillary channels to ionizethe proteins. The translational stage will be moved to consecutivelybring the whole open channel to the focused laser spot. Therefore,2-dimensional protein separation and analysis can be achieved. Theprotein molecular ions from the MALDI source can be fragmented by postsource decay or collisionally activated dissociation for proteinidentification.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in bioanalytical chemistry,or molecular biology, or related fields are intended to be within thescope of the claims.

1. An isoelectric focussing (IEF) module comprising: (a) a first planarmember having a covered channel along which a sample may be loaded andat least one compound compount thereof focussed isoelectrically; and (b)means for exposing the channel along at least a portion of its lengthand thereby exposing the sample or component(s) therewithin.
 2. A moduleaccording to claim 1, in which the sample or component are accessiblealong the exposed channel at substantially the positions at which theyare focussed.
 3. A module according to claim 1, in which the channelcomprises an open channel which is exposed along at least a portion ofits length.
 4. A module according to claim 1, in which the channelcomprises a linear groove formed on a surface of the first planarmember.
 5. A module according to claim 1, in which the channel comprisesa microchannel or capillary channel.
 6. A module according to claim 5,in which the microchannel or capillary channel is microfabricated on thefirst planar member.
 7. A module according to claim 1, in which thechannel has a width of between 1 to 500 micrometres, more preferablybetween 50 to 350 micrometres, more preferably between 50 to 350micrometres, most preferably about 150 micrometers or about 175micrometres.
 8. A module according to claim 1, in which the first planarmember is formed from a material selected from the group consisting of:plastics, polymers, ceramic, glass or composite.
 9. A module accordingto claim 1, in which at least one wall of the channel comprisespoly(methylmethacrylate) (PMMA) or polycarbonate.
 10. A module accordingto any of claim 1, in which the first planar member is coated orderivatised to reduce surface charge and thereby minimise electroosmoticflow (EOF).
 11. A module according to claim 1 further comprising areservoir for electrolyte, the reservoir being in electrical connectionwith the channel.
 12. A module according to claim 11, in which thereservoir is formed on the first planar member adjacent to each end ofthe channel.
 13. A module according to claim 1, further comprising a lidbeing a second planar member, which reduces evaporation of the sample inthe channel.
 14. A module according to claim 13, in which the lidcomprises an elongate recess on its inner face, the recess beingpositioned such that when the lid is mated with the first planar member,no substantial leakage of sample contained in the channel occurs.
 15. Amodule according to claim 14, in which the length and width of therecess are at least as great as a channel in the first planar member.16. A module according to claim 11 and 13, in which the reservoir isdisposed on the lid.
 17. A module according to claim 11, furthercomprising means for electrical connection between the channel and thereservoir without resulting in substantial mixing of sample andelectrolyte.
 18. A module according to claim 17, in which said meanscomprises a semi-permeable membrane, agarose, or a gel plug.
 19. Amodule according to claim 1, comprising which comprises a plurality ofchannels in substantially parallel orientation.
 20. A module accordingto claims 1 and 19, in which a channel comprises a closed channel andthe means for exposing the channel comprises lines of weakness enablingfracture along a longitudinal plane of the channel.
 21. A moduleaccording to claim 1 further comprising a translational stage on whichis mounted the first planar member.
 22. An apparatus for separating oneor more components in a sample, the apparatus comprising an isoelectricfocussing module according to claim 1, together with a module capable ofseparating isoelectrically focussed components according to theirrespective masses.
 23. An apparatus according to claim 22, in which themass separation module comprises a module for mass spectrometry.
 24. Anapparatus according to claim 23 in which the mass separation modulecomprises a matrix assisted laser desorption/ionisation massspectrometry (MALDI-MS) module.
 25. A method of separating one or morecomponents in a sample, the method comprising the steps of: (a)providing an isoelectric focussing (IEF) module comprising a firstplanar member having a covered channel; (b) loading the covered channelwith a sample; (c) isoelectrically focussing a component or componentsof the sample along the channel; (d) exposing the channel along at leasta portion of its length and thereby exposing the sample or component(s)therewithin; (e) optionally coating the isoelectric focussing (IEF)module with an electrical conductive thin film or layer; and (f)optionally analysing one or more separated components by massspectrometry.
 26. (canceled)
 27. A method according to claim 25, furthercomprising the step of: (e) accessing one or more components in the openchannel and analysing it.
 28. A method according to claim 27, in whichthe or each component is analysed by mass spectrometry.
 29. A methodaccording to claim 25, in which the sample comprises a MALDI matrix. 30.A method according to claim 29, in which a MALDI matrix is added to thesample subsequent to isoelectric focussing.
 31. A method according toclaim 29 or 30, in which the MALDI matrix is selected from the groupconsisting of: Cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxybenzoic acid (DHB), Alpha CCA, Sinapinic Acid (SA), 3-hydroxypicolinicacid (HPA), IAA (Na⁺), 2-(4-Hydroxyphenylazo)benzoic acid HABA (Na⁺),Dithranol (Na⁺), Retinoic Acid (Na⁺), Succinic acid,2,6-Dihydroxyacetophenone, Ferulic Acid, Caffeic acid, Glycerol and4-Nitroaniline.
 32. A method of analysing a molecule, the methodcomprising: (a) providing an elongate open channel; (b) introducing aplurality of molecules into the elongate open channel; (c) separatingmolecules along the elongate open channel according to their isoelectricpoints; and (d) accessing a molecule in the elongate open channel andanalysing it.
 33. An apparatus for isoelectric focussing (IEF) ofmolecules, the apparatus comprising an elongate channel, in which theelongate channel is open along at least a portion of its length toenable separated molecules to be accessed.
 34. (canceled)
 35. A kitcomprising a module or apparatus as claimed in claim 1, 22 and 33 andfurther comprising a sample comprising proteins to be analysed.
 36. Amethod of detecting a protein in a sample, the method comprising thesteps of: (a) providing an elongate open channel; (b) introducing thesample into the elongate open channel; (c) separating the protein alongthe elongate open channel according to its isoelectric point; (d)accessing the protein in the elongate open channel; and (e) detectingthe protein by mass spectrometry.
 37. (canceled)
 38. (canceled) 39.(canceled)
 40. (canceled)
 41. A method of diagnosis of a disease in anindividual, the method comprising the steps of: (a) providing a cell,tissue or organ sample from an individual, and producing a proteincontaining extract thereof; (b) detecting a disease associated proteinin the sample using a module according to claim 1, an apparatusaccording to claim 22, or a method according to claim
 25. 42. A meansfor interfacing a capillary isoelectric focussing (CIEF) apparatus and aMALDI-MS apparatus comprising a covered channel along which isoelectricfocussing is carried out, and means for exposing said channel along atleast a portion of its length and thereby exposing the sample orcomponent(s) therewithin.
 43. An interface according to claim 42 whichcomprises an open channel.
 44. An apparatus according to claim 24wherein the mass separation module comprises a matrix assisted laserdesorption/ionization-time of flight (MALDI-TOF).
 45. The methodaccording to claim 25 wherein the components are analyzed by MALDI-MS.46. The method according to claim 25 wherein the components are analyzedby MALDI-TOF.
 47. The method according to claim 27 wherein a componentis analyzed by MALDI-MS.
 48. The method according to claim 27 wherein acomponent is analyzed by MALDI-TOF.
 49. A means for interfacing acapillary isoelectric focussing (CIEF) apparatus and a MALDI-TOFapparatus comprising a covered channel along which isoelectric focussingis carried out, and means for exposing said channel along at least aportion of its length and thereby exposing the sample or component(s)therewithin.
 50. An interface according to claim 43 which comprises anopen channel.